Response to comment MCN‐09‐21‐LE‐5028—Human milk sampling should be standardised if the adequacy of human milk nutrients is assessed

Dear Editor,

We thank Dr. Bzikowska‐Jura for the interest in our work and for the comment provided. We could not agree more that the conditions of both human milk sampling and sample handling and storage are often of great importance for results on milk composition, and hence we fully support the conclusion that respective studies need to be thoughtfully planned and meticulously implemented. In fact, we have previously emphasised the need for representative human milk sampling in research studies (Demmelmair et al., 2020; Demmelmair & Koletzko, 2018; Jensen et al., 1997). The concentrations of different nutrients in human milk are associated with variables such as feeding practice, time of the day, stage of lactation, degree of breast expression, and others. Regarding iron concentration in human milk, a recent systematic review identified consistent indications for circadian variation, with iron concentration tending to increase during the night with a peak of milk iron in the morning, and a decrease during the day to a nadir around 18 h (Italianer et al., 2020). A study in Guatemala also reported appreciable day‐to‐day variation in human milk iron but not in zinc concentrations in milk collected from women at the same time of the day for 3–4 consecutive days (Dhonukshe‐Rutten et al., 2005). In contrast to iron, there is no conclusive evidence of diurnal variation of human milk zinc concentration.

In our study (Dumrongwongsiri et al., 2021) we tried to account for these potential influencing factors. We performed human milk collection from all participants in the mornings, with a maximum allowed time window from 9.30 to 12.00 h. We did not ask the participating breastfeeding women participants to fast before milk collection and did not wish to interfere with their breastfeeding practice, resulting in different time intervals to the preceding breastfeeding occasion. We took this choice to respect the needs of infants and mothers and to avoid undue disturbance of breastfeeding. Moreover, thereby our results reflect the real‐life situation if iron and zinc supply to breastfed infants better than by using a somewhat artificial study design with a strictly defined time interval to the last feed. Women emptied one breast as completely as possible using a hospital‐grade, electric milk pump (Medela Lactina; Medela). All equipment and containers used for milk collection were treated with a nitric acid solution to prevent trace element contamination. The collected milk collection was thoroughly mixed and an aliquot of 10–15 ml was transferred to plastic tubes and frozen at −80°C.

Collecting all human milk of lactating women over a 24‐h period may provide more accurate data on the total nutrient supply to the breastfed infant, but it raises ethical concerns as it implies that infants cannot be breastfed on study days but will have to be given pumped milk by bottle, cup, or spoon‐feeding. In our view, one should very carefully balance whether the added considerable burden and disadvantages for the study subjects are justified by an added gain of relevant information, and this would have to be evaluated by an independent ethical committee. Based on data of the study performed in Guatemala (Dhonukshe‐Rutten et al., 2005) it was proposed that evaluation of two human samples collected on consecutive days may serve to accurately assess a mother's breast milk iron and copper concentrations, for example, for determining the relationship of breast milk iron concentration with maternal diet or maternal status or to assess the impact of interventions (de Pee & Hautvast, 2005). Considering the reported diurnal variation of milk iron concentration (Italianer et al., 2020) another potential approach could be to collect two or more samples of human milk at different times of a day in an effort to better reflect the circadian variation of milk nutrient concentrations. Future studies may benefit from the exploration of different protocols and strategies for human milk collections and their value for providing accurate and representative information on nutrient supply to breastfed infants.

We believe that our study provides valuable and novel, quantitative data on the total intake of the critical micronutrients iron and zinc in a sizeable group of breastfed infants in a developing country, based on prospective study design with a strictly standardised study protocol that includes measuring not on only milk composition but also measuring the volumes of infant milk intakes.